This proposal will fill a gap in our understanding of key events in secretion, namely, the nature of changes in plasma membrane morphology and biochemistry during the coupled processes of exocytosis and endocytosis. When triggered by Ca2+, neuroendocrine cells release biogenic amines and neuropeptides into the extracellular space through fusion of dense core granules with the plasma membrane. Granule membranes are then retrieved for reuse via endocytosis. A precisely orchestrated series of events underlie the proper execution of these tasks. Prior to exocytosis, biochemical interactions between granule and plasma membrane proteins and lipids occur priming the granule for fusion;the inositol phospholipid Pl(4,5) P2 is critical for these events. The respective membranes are then ruptured and merged to form a fusion pore. After exocytosis, other interactions occur to deform the plasma membrane and recover granule membrane constituents. In recent years, a cohort of proteins thought to be important in exo-/endocytosis has been identified. Some of these are predicted to alter membrane morphology, either prior to exocytosis, such as synaptotagmin, or after exocytosis, such as endophilin and amphiphysin. The timing and the location of these changes may be influenced by their interaction with PI(4,5)P2. There is also uncertainty about the length of time the granule membrane retains its curvature before it flattens into the plasma membrane or is retrieved by endocytosis. Because rapid, high resolution detection of plasma membrane events has been difficult, the elucidation of these processes is still lacking. The unifying hypothesis behind this proposal is that the underlying biochemical and physiological changes in the plasma membrane important for secretion result in changes of membrane conformation. The Specific Aims are designed to: 1) Determine the morphological dynamics of the plasma membrane associated with exocytosis and endocytosis;2) Determine the role of PI(4,5)P2 in regulating properties of the plasma membrane important for secretion. The experiments will utilize adrenal chromaffin cells, and confocal and TIR-based optical methods. It is anticipated that the results of our studies will provide fundamental insights into the regulation and dynamics of the secretory response. They will also have broad relevance based on the similarity of the chromaffin cell model to other neuroendocrine cells and to presynaptic pathways in neurons. A more complete understanding of the mechanisms underlying exo-/endocytosis will help drive the development of rational strategies for the treatment of cardiovascular, endocrine and neurological diseases.